Methods for protein labeling based on acyl carrier protein

ABSTRACT

A method for labeling acyl carrier protein (ACP) fusion proteins with a wide variety of different labels is disclosed. The method relies on the transfer of a label from a coenzyme A type substrate to an ACP fusion protein using a holo-acyl carrier protein synthase (ACPS) or a homologue thereof. The method allows detecting and manipulating the fusion protein, both in vitro and in vivo, by attaching molecules to the fusion proteins that introduce a new physical or chemical property to the fusion protein. Examples of such labels are, among others, spectroscopic probes or reporter molecules, affinity tags, molecules generating reactive radicals, cross-linkers, ligands mediating protein-protein interactions or molecules suitable for the immobilization of the fusion protein.

This application is a divisional of Ser. No. 10/557,897, filed Nov. 22,2005 now U.S. Pat. No. 7,666,612, which is a 371 U.S. national stage ofInternational Application No. PCT/IB2004/001733 filed May 19, 2004,herein incorporated by reference in their entirety.

FIELD OF THE INVENTION

The present invention relates to methods of transferring a label from asubstrate to a fusion protein comprising a protein of interest and anacyl carrier protein or a fragment thereof, and in particular to methodswhich further comprise detecting and/or manipulating the labeled fusionprotein.

BACKGROUND OF THE INVENTION

Progress in understanding complex biological systems depends oncharacterizing the underlying interactions of biomolecules, inparticular proteins. While the DNA sequencing of an increasing number oforganisms has identified their open reading frames (ORF), thepossibilities to characterize the corresponding proteins in vivo and invitro are limited. Most strategies that aim at realizing this objectiveare based on the construction of a fusion protein that either allows thepurification of the fusion for in vitro applications or allows followingthe protein in vivo. Examples for such tags include the 6xHis tag,glutathione S transferase, maltose binding protein, epitope tags,yeast-two hybrid system, O⁶-alkylguanine-DNA alkyltransferase,split-ubiquitin, and green fluorescent protein (GFP) fusion proteins.However, all these techniques have various limitations or disadvantages.

Gehring et al. (1997) and Lambalot and Walsh (1995) describe the use ofE. coli holo acyl carrier protein synthase (ACPS) to catalyze theposttranslational modification of apo-acyl carrier protein (apo-ACP) byattaching the cofactor 4′-phosphopantetheine (P-pant) to a conservedserine residue in vitro, yielding holo-ACP. The source of P-pant iscoenzyme A. Gehring et al. (1997) demonstrate furthermore that, by usingcoenzyme A analogs, which are modified in the P-pant part but still ableto serve as substrates for ACPS, holo-ACP's with modified P-pants ascofactor are obtained.

Isolated phosphopantetheinyl transferases such as ACPS are described inInternational Patent Application WO 97/13845.

A method of transferring a label to O⁶-alkylguanine-DNA alkyltransferase(AGT) fusion proteins, and the use of this method for the detection ofAGT fusion proteins is described in International Patent Application WO02/083937.

SUMMARY OF THE INVENTION

The invention relates to a method for detecting and/or manipulating aprotein of interest, which comprises contacting a fusion proteincomprising protein of interest and an acyl carrier protein (ACP) or afragment thereof with a labeled coenzyme A (CoA) type substrate and aholo-acyl carrier protein synthase (ACPS) or a homologue thereof so thatthe ACPS transfers the label to the fusion protein, and optionallydetecting and/or further manipulating the labeled fusion proteinobtained, using the label in a system designed for recognising and/orhandling the label.

Furthermore the invention relates to the use of a fusion proteincomprising protein of interest and an ACP or a fragment thereof in sucha method. In particular, the method of the invention is used forpurifying or immobilizing a protein of interest, or for continuouslymonitoring a protein of interest in vitro or in vivo due to the labelattached to it in the method of the invention.

The protein of interest incorporated into the fusion protein of theinvention may be of any kind, which includes proteins, polypeptides andpeptides of any length and both with and without secondary, tertiary orquaternary structure.

The particular labeled coenzyme A (CoA) type substrates used in themethod of the invention are obtainable from CoA or modified CoA byattachment of a linker with at least one reactive site for furtherattachment of a label, i.e. a detectable marker. The invention alsorelates to such novel labeled coenzyme A (CoA) type substrates, tomethods of manufacture thereof, to intermediates useful in the synthesisof such novel CoA type substrates, and to their use in the method of theinvention.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1:

(A) Analysis of the reaction of 6xHis-ACP (1 μM), 6xHis-ACPS (0.2 μM)and CoA-Bt (5 μM). At indicated times (t) aliquots are removed from thereaction mixture and biotinylation of 6xHis-ACP probed by Westernblotting using a streptavidin-peroxidase conjugate.

(B) Quantification of biotinylation of 6xHis-ACP: 6xHis-ACP (3 μM),6xHisACPS (5 μM) and CoA-Bt (10 μM) are incubated for 30 min, dialyzeand aliquots of the sample are incubated with streptavidin and appliedto SDS-PAGE (lane No. 2). The formation of a stable biotin-streptavidincomplex leads to a gel shift of biotinylated protein. The amount ofbiotinylation is estimated by comparing the band intensity of 6xHis-ACPin lane No. 2 with that of a sample containing identical concentrationsof 6xHis-ACP but no streptavidin (lane No. 1).

(C) Competition assay between CoA and CoA-Bt or CoA-Dg as ACPSsubstrates: 6xHis-ACP (0.4 μM), 6xHis-ACPS (0.4 μM), CoA-Bt or CoA-Dg (2μM) and varying concentrations of CoA (0-80 μM) are incubated for 30 minand the degree of labeling determined by Western blotting. The relativesignal intensities (I) in the Western blots are plotted against[CoA]/[CoA-L](L=label) and fitted to the equation I=A/(1+Bx), where xrepresents [CoA]/[CoA-L], A an unspecified constant and B the ratio(k_(cat)/K_(M))_(CoA)/(k_(cat)/K_(M))_(CoA-L). These experiments yieldratios of the specificity constants of 0.48 for(k_(cat)/K_(M))_(CoA)/(k_(cat)/K_(M))_(CoA-Bt) and of 0.35 for(k_(cat)/K_(M))_(CoA)/(k_(cat)/K_(M))_(CoA-Dg), revealing no significantdiscrimination between free and derivatized CoA.

FIG. 2

(A-F) Labeling of ACP fusion proteins on cell surfaces of yeast.Fluorescence micrographs of yeast cells expressing Aga2-ACP which arelabeled with either Cy3 (A) or biotin followed by streptavidin-coatedquantum dots (D). (B) as (A) and (E) as (D) but with cells notexpressing AGA2-ACP: (C) and (F) are the transmission micrograph of thesame samples as in (B) and (D), respectively. These experiments indicatethat only the yeast cells expressing Aga2p-ACP are labelled.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method for detecting and/or manipulating aprotein of interest, which comprises contacting a fusion proteincomprising protein of interest and an acyl carrier protein (ACP) or afragment thereof with a labeled coenzyme A (CoA) type substrate and aholo-acyl carrier protein synthase (ACPS) or a homologue thereof so thatthe ACPS transfers the label to the fusion protein, and optionallydetecting and/or further manipulating the labeled fusion proteinobtained using the label in a system designed for recognising and/orhandling the label.

ACP or ACP domains act as carriers in the biosynthesis of fatty acids,polyketides and in non-ribosomal peptide synthesis. ACP isposttranslationally modified by holo-acyl carrier protein synthase(ACPS) which transfers the 4-phosphopantetheine cofactor from thecoenzyme A to a conserved serine residue of ACP. ACPSs are also calledphosphopante-theinyl transferases. It has been shown that ACPSspossesses a relatively low substrate specificity concerningmodifications of CoA at the thiol group of the phosphopantetheinylmoiety of CoA. Taking advantage of this, ACP fusion proteins can bespecifically labeled by incubation with ACPS and a CoA derivative thatcarries the label via the phospho-pantetheinyl moiety. The label is thustransferred with the phosphopantetheinyl moiety to the conserved serineresidue of the ACP. The labelling is independent of the nature of thefusion protein.

The terms ACP and ACPS here stand for any pair of proteins in which oneof the two (the ACP) is an acceptor for a phosphopantetheinyl derivativethat originates from a CoA derivative and the other one (the ACPS)catalyzes the transfer of the phosphopantetheinyl derivative to thisACP.

The terms ACPS and phosphopantetheinyl transferase are usedinterchangeably herein despite the fact that a number ofphosphopantetheinyl transferases homologous to ACPS from E. coli modifyproteins that do not participate in fatty acid synthesis but rather inthe biosynthesis of natural products (Lambalot et al., 1996). Examplesfor such phospho-pantetheinyl transferases are: EntD, participating inenterobactin synthesis; Sfp and Psf-1, participating in surfactinbiosynthesis; Gsp, participating in gramidicin S biosynthesis; LYS5,participating in lysine biosynthesis; Bli, participating in bacitracinbiosynthesis; Lpa-14, participating in iturin A biosynthesis; and NshC,participating in nosiheptide biosynthesis. This invention includes theuse of all ACPSs in the labeling scheme that are homologous to the ACPSfrom E. coli.

The term ACP stands for any protein that will be posttranslationallyphosphopante-theinylated by ACPS from E. coli or any ACPS homologous tothe ACPS from E. coli as defined by Lambalot et al. (1996). Thisincludes proteins not only participating in fatty acid synthesis butalso in polyketide synthesis, non-ribosomal peptide synthesis, aminoacid synthesis and depsipeptide synthesis. In addition to theposttranslational modification by ACPS these proteins have also incommon that they form acyl-pantetheinyl thiolesters with differentsubstrates and that the phosphopantetheinyl moiety is attached to aserine residue. In their natural function the ACPs might be the domainof a multi-functional enzyme (as in type I fatty acid synthases) or aseparate protein (as in type II fatty acid synthases).

“Detecting” means observing the label and the protein of interestattached to it based on the properties of a label in a system designedfor observing the label, and includes recognising the particular label,finding the label in a particular environment due to the properties ofthe label, optionally quantifying the label and the protein attached toit, and optionally determining the properties of the micro-environmentof the label, and the like. “Manipulating” means handling the label andthe protein of interest attached to it, and includes handling the labeland the protein of interest attached to it based on the properties of alabel in system designed for handling the label, and includes separatingfrom a chemical or biological environment, introducing into anotherchemical or biological environment, purifying, i.e. separating fromunwanted side products and impurities, immobilizing by reaction of thelabel with a solid carrier, contacting with chemical or biologicalreagents so as to modify the properties of the label and/or of theprotein of interest, and the like.

The method disclosed herein is generally applicable to a range ofapplications and is capable of specifically and covalently labelingfusion proteins with (1) labels which are capable of sensing andinducing changes in the environment of the labeled fusion protein, (2)labels which aid in manipulating the fusion protein by the physicaland/or chemical properties specifically introduced by the label to thefusion protein and/or (3) labels which aid in purification of the fusionprotein through the properties introduced by the label. The methoddisclosed herein can be used to label ACP fusion proteins both in vitroand in vivo, e.g. in cells.

Furthermore the invention relates to the use of a fusion proteincomprising protein of interest and an ACP or a fragment thereof in sucha method. In particular, the method of the invention is used forpurifying or immobilizing a protein of interest, or for continuouslymonitoring a protein of interest in vitro or in vivo due to the labelattached to it in the method of the invention.

In one aspect, the present invention provides a method for continuouslymonitoring a protein of interest in vitro or in vivo, wherein a fusionprotein comprising protein of interest and an acyl carrier protein (ACP)or a fragment thereof is contacted with a labeled coenzyme A (CoA) typesubstrate and a holo-acyl carrier protein synthase (ACPS) or a homologuethereof so that the ACPS transfers the label to the fusion protein, andthe label is observed in a system designed for recognising the label.

In a further aspect, the present invention provides a method formanipulating a protein of interest in vitro or in vivo, wherein a fusionprotein comprising protein of interest and an acyl carrier protein (ACP)or a fragment thereof is contacted with a labeled coenzyme A (CoA) typesubstrate and a holo-acyl carrier protein synthase (ACPS) or a homologuethereof so that the ACPS transfers the label to the fusion protein, andthe fusion protein is manipulated based on the physical and/or chemicalproperties of the label.

In a particular aspect, the physical and/or chemical properties of thelabel allow efficient purification of the labeled fusion protein, andthe present invention accordingly provides a method of purifying aprotein of interest by performing the steps of the method of theinvention, using the physical and/or chemical properties of the labelfor purification, and cleaving the fusion protein thereafter providingpure protein of interest.

In a further aspect, the present invention provides a method ofimmobilizing a fusion protein comprising protein of interest and an acylcarrier protein (ACP) or a fragment thereof on a solid support, themethod comprising contacting the fusion protein with a labeled coenzymeA (CoA) type substrate which is attached or attachable to a solidsupport, wherein the holo-acyl carrier protein synthase (ACPS) transfersthe label so that it is covalently bonded to the ACP fusion proteinwhich thereby is attached or can be subsequently attached to the solidsupport. In particular embodiments of the invention in which the labelis not initially attached to the solid support, the method may involvethe further step of contacting the labeled ACP fusion protein with thesolid support so that it becomes immobilized on the solid support. Inthese preferred embodiments of the invention, the label may becovalently attached to the solid support, either when the label istransferred or in a subsequent reaction, or may be one member of aspecific binding pair, the other member of which is attached orattachable to the solid support, either covalently or by any othermeans, e.g. using the specific binding pair of biotin and avidin orstreptavidin.

In a further aspect, the present invention provides a method to labelACP fusion proteins both in vivo as well as in vitro. The term in vivolabeling of an ACP fusion protein includes labeling in all compartmentsof a cell as well as of ACP fusion proteins pointing to theextracellular space. If the labeling of the ACP fusion protein is donein vivo and the protein fused to the ACP is a plasma membrane protein,the ACP part of the fusion protein can be either attached to thecytoplasmic or the extracellular side of the plasma membrane. If thelabeling is done in vitro, the labeling of the fusion protein can beeither performed in cell extracts or with purified or enriched forms ofthe ACP fusion protein.

The present invention is based on the realization that specificattachment of a label to a desired protein could be carried out byconstructing a fusion protein between that protein of interest and theacyl carrier protein (ACP) or a fragment thereof.

In a preferred application, the acyl carrier protein or “ACP” has theproperty of being modified by the holo-acyl carrier protein synthase or“ACPS” in a way that labeled 4′-phosphopantetheine is transferred fromthe appropriate coenzyme A (“CoA”) to a serine residue of ACP or afragment thereof forming part of the fusion protein. In preferredembodiments, ACP is, for example, E. coli acyl carrier protein which isdescribed in Rawling and Cronan, 1992, and references therein. However,other acyl carrier proteins (ACP) are known and may be used in theinvention, e.g. ACP from Streptomyces species described in Gehring etal., 1997, or any ACP having the property of being modified by ACPSdefined above in the presence of appropriately labeled CoA. In thepresent invention, ACP also includes variants of a wild-type ACP whichmay differ by virtue of one or more amino acid substitutions, deletionsor additions, but which still retain the property of serving as anacceptor for labeled 4′-phosphopantetheine in the reaction catalyzed byACPS. Other variants of ACP may be chemically modified using techniqueswell known to those skilled in the art. ACP variants may be producedusing protein engineering techniques known to the skilled person and/orusing molecular evolution to generate and select new acceptor sequencesfor transfer of labeled 4′-phosphopantetheine in the reaction catalyzedby ACPS. ACP fragments are those which contain the serine residue towhich the phosphopantetheine derivative is attached, and which retainthe function to accept such phosphopantetheine derivative.

In preferred embodiments, the ACPS is, for example, E. coli holo-acylcarrier protein synthase which is described in Lambalot and Walsh, 1995,and references therein. However, other holo-acyl carrier proteinsynthases are known, such as ACPS from Bacillus subtilis described inLambalot et al., 1996, or any form of the protein which can be employedin the present invention provided that they have the property ofmodifying ACP defined above in the presence of appropriately labeledCoA. These ACPSs are also known as phosphopantetheinyl transferases asdescribed in Lambalot et al., 1996, and the present invention includesthe use of this general class of enzymes. In the present invention,holo-acyl carrier protein synthases also includes variants of awild-type ACPS which may differ by virtue of one or more amino acidsubstitutions, deletions or additions, but which still retain theproperty of transferring labeled 4′-phosphopantetheine specifically tothe ACP fusion protein. Other variants of ACPS may be chemicallymodified using techniques well known to those skilled in the art. ACPSvariants may be produced using protein engineering techniques known tothe skilled person and/or using molecular evolution to generate andselect new specificities for transfer of labeled 4′-phosphopantetheineto different acceptor sequences.

For the labeling of ACP fusion proteins by ACPS a number ofconsiderations are advantageously taken into account. Most importantly,the labeling of the ACP fusion protein relies on the presence of the ACPpart of the ACP fusion protein in its apo form before labeling. If theACP fusion protein is expressed in a host that possesses an endogenousACPS that accepts this ACP fusion protein as a substrate forposttranslational modification the ACP fusion protein can be at leastpartially blocked for the desired modification. To minimize thisunwanted modification of ACP fusion proteins different solutions areproposed. Firstly, an ACP is chosen which is not efficiently modified bythe ACPS of the host, i.e. the ACP is orthogonal to the biochemistry ofthe host. For example, the ACP from E. coli is not modified to asignificant extent by the human ACPSs when expressed as a fusion proteinin human cells (see FIG. 3). Secondly, overexpression of the ACP fusionprotein will lead to the predominant formation of apo-ACP in ACP fusionproteins. For example, overexpression of 6xHis-ACP from E. coli in E.coli leads to the formation of mostly apoLACP in 6xHis-ACP, although theendogenous ACPS is present in these cells.

For the labeling reaction, ACP fusion proteins have also to be contactedwith both the corresponding ACPS and the CoA derivative. This impliesthat for in vivo applications the ACP fusion protein is either presentedon the surface of the cell or the ACPS and the CoA derivative areintroduced into the cell of interest using techniques such asmicroinjection.

In the present invention, the reference to the protein part of thefusion protein with the ACP is intended to include proteins,polypeptides and peptides of any length and both with and withoutsecondary, tertiary or quaternary structure, and preferably consists ofat least twelve amino acids and up to 2000 amino acids, preferablybetween 50 and 1000 amino acids. The protein of interest according tothe invention is selected from the group consisting of enzymes,DNA-binding proteins, transcription regulating proteins, membraneproteins, nuclear receptor proteins, nuclear localization signalproteins, protein cofactors, antibodies, membrane pump proteins,membrane channel proteins, membrane carrier proteins, motor proteins,proteins involved in signal transduction, nuclear proteins, ribosomalproteins, small monomeric GTPases, ATP-binding cassette proteins,intracellular structural proteins, proteins with sequences responsiblefor targeting proteins to particular cellular compartments, proteinsgenerally used as labels or affinity tags, and domains or subdomains ofthe aforementioned proteins. The ACP fusion protein may consist of oneor more, e.g. one, two or three, proteins of interest fused to ACP atthe N-, C- or N- and C-terminal of ACP.

More particularly, the protein of interest according to the invention isselected from the group consisting of

-   enzymes, e.g. transferases (EC 2), more specific a transferase    transferring an alkyl or aryl group other than a methyl group (EC    2.5), in particular a glutathione transferase (EC 2.5.1.18),-   or a kinase, that is a transferase transferring phosphorus    containing groups (EC 2.7), in particular a kinase with an alcohol    group as acceptor (EC 2.7.1), such as a protein kinase with serine    and threonine as the phosphorylated target sites in the substrate    protein, e.g. casein kinase from yeast (EC 2.7.1.37), or a tyrosine    protein kinase (EC 2.7.1.112); or e.g. oxidoreductases (EC 1), more    specific an oxidoreductase acting on peroxide as acceptor (EC 1.11),    in particular the enzyme cytochrome C peroxidase (EC 1.11.1.5); or    e.g. hydrolases (EC 3), more specific a hydrolase acting on an ester    bond (EC 3.1), in particular a phosphoric monoester hydrolase (EC    3.1.3), such as a protein phosphoric monoester hydrolase; or a    hydrolase hydrolysing peptide bonds, also known as peptidase or    protease (EC 3.4), in particular a caspase;-   DNA-binding proteins, more specific transcription repressor proteins    which are protein factors inhibiting mRNA synthesis, specifically a    protein factor inhibiting mRNA synthesis in E. coli, in particular    the DNA-binding domain of the LexA protein;    transcription regulating proteins, more specific transcription    repressor proteins, in particular transcription repressor proteins    containing a tryptophan/aspartate repeat structure,-   membrane proteins, e.g. membrane proteins showing at least one    transmembrane helix, more specific membrane proteins from the    endoplasmatic reticulum (ER) membrane, in particular membrane    proteins being active in protein translocation into the ER, such as    the ER transmembrane protein Sec62;-   or e.g. a protein from the family of 7-transmembrane helix (7-TM)    proteins, more specific a 7-TM protein being a G-protein coupled    receptor (GPCR), in particular those that bind macromolecular    ligands with a molecular weight above 1 kDa, such as a mammalian,    e.g. human, neurokinin-1-receptor (NK1);-   or e.g. transmembrane ion channel proteins from the cell membrane,    in particular ligand gated ion channel proteins, more specific a    ligand gated ion channel proteins sensitive to serotonin, such as    the serotonin receptor 5-HT3;-   or e.g. membrane receptors other than ion channels and G-protein    coupled receptors; or e.g. peroxisomal membrane proteins, in    particular from yeast, such as the protein Pex15;-   nuclear receptor proteins, e.g. nuclear receptor proteins from the    family of transcription factors, more specific nuclear receptor    proteins from the family of ligand inducible transcription factors,    in particular a nuclear receptor from the family of steroid, e.g.    estrogen, receptors, such as the human estrogen receptor hER;-   nuclear localization signal proteins, such as the nuclear    localization signal from the Simian Virus 40 (SV40);-   protein cofactors, e.g. proteins containing an ubiquitin sequence in    their genetic structure; small monomeric GTPases, more specific    membrane-adherent small monomeric GTPases, e.g. a member of the Ras    family;-   ATP-binding cassette (ABC) proteins, e.g. a multiple drug resistance    protein; intracellular structural proteins, more specifically    proteins of the cytoskeleton, more specifically human cytoplasmic    β-actin;-   proteins with sequences responsible for targeting proteins to    particular cellular compartments, e.g. to the Golgi apparatus, the    endoplasmatic reticulum (ER), the mitochondria, the plasma membrane    or the peroxisome;-   proteins generally used as labels or affinity tags, e.g. fluorescent    proteins giving a fluorescent signal on excitation with UV or    visible radiation, in particular fluorescent proteins from the    family known as green fluorescent proteins (GFP), such as the    fluorescent protein known as enhanced cyano fluorescent protein    (ECFP);    and domains or subdomains of the aforementioned proteins.

Furthermore, the protein of interest according to the invention isselected according to source. In particular, proteins of interest arethose present in human species, mice, rats, other higher mammals,eukaryotic species, bacterial species, e.g. salmonella, more specificsalmonella typhi or salmonella typhimurium, mycobacteria, more specificmycobacterium tuberculensis, or staphylococci, more specificstaphylococcus aureus, or from a viral source, e.g. humanimmunodeficiency virus (HIV), human influenza virus, hepatitis virus, orcorona viruses.

Furthermore, the protein of interest is selected according to its rolein a certain disease, such as cancer, cardiovascular diseases, mentaldisorders, Alzheimer, obesity, viral infections, and bacterialinfections.

In a particular embodiment of the invention the fusion proteins are madefrom E. coli ACP or variants of such ACP DNA on the one side andproteins of interest (as listed above) encoding sequences eitherattached to the N-terminal (N) or the C-terminal (C) side or N- andC-terminal side of the ACP DNA sequence, leading to the fusion proteinsof the invention. Fusion proteins may further contain suitable linkers,e.g. linkers which may be susceptible to enzyme cleavage under suitableconditions, between ACP and the protein of interest and/or between twoproteins of interest in a fusion protein. Examples of such linkers arethose which are cleavable at the DNA stage by suitable restrictionenzymes, e.g. AGATCT cleavable by Bgl II, and/or linkers cleavable bysuitable enzymes at the protein stage, e.g. tobacco etch virus NIa (TEV)protease.

Fusion proteins may be expressed in prokaryotic hosts, preferably E.coli, or eukaryotic hosts, e.g. yeast, fungal, insect or mammaliancells.

The invention also relates to novel fusion protein comprising a proteinof interest and an ACP or a fragment thereof.

In the present invention, the labeled CoA type substrate is preferably alabeled coenzyme A derivative possessing the following general formula(I):

or the following general formula (II):

whereinR is a linker group bridging the coenzyme A and the label; and “Label”is a label molecule suitable for the detection, purification and/ormanipulation of the fusion protein as described herein.

However, the invention is not restricted to the substrates of formula(I) or (II) since a wide range of other substrates can be used for atransfer of a label to ACP fusion proteins. For example, substitutionsof the purine part, modifications of the sugar moiety or of thepanthetheinylic acid moiety of a compound of formula (I) are considered.

A linker group R in a compound of formula (I) or (II) is a flexiblelinker connecting a label to the coenzyme A. Linker units are chosen inthe context of the envisioned application, i.e. in the transfer of thelabel to a fusion protein comprising ACP. They also increase thesolubility of the substrate in the appropriate solvent. The linkers usedare chemically stable under the conditions of the actual application.The linker R does not interfere with the reaction with ACP nor with thedetection of the label, but may be constructed such as to be cleaved atsome point in time after the reaction of the coenzyme A type substratewith the fusion protein comprising ACP.

A linker group R is a straight or branched chain alkylene group with 1to 300 carbon atoms, wherein optionally

-   (a) one or more carbon atoms are replaced by oxygen, in particular    wherein every third carbon atom is replaced by oxygen, e.g. a    poylethylenoxy group with 1 to 100 ethylenoxy units;-   (b) one or more carbon atoms are replaced by nitrogen carrying a    hydrogen atom, and the adjacent carbon atoms are substituted by oxo,    representing an amide function —NH—CO—;-   (c) one or more carbon atoms are replaced by oxygen, and the    adjacent carbon atoms are substituted by oxo, representing an ester    function —O—CO—;-   (d) the bond between two adjacent carbon atoms is a double or a    triple bond, representing a function —CH═CH— or —C≡C—;-   (e) one or more carbon atoms are replaced by a phenylene, a    saturated or unsaturated cycloalkylene, a saturated or unsaturated    bicycloakylene, a bridging heteraromatic or a bridging saturated or    unsaturated heterocyclyl group;-   (f) two adjacent carbon atoms are replaced by a disulfide linkage    —S—S—;    or a combination of two or more, especially two, alkylene and/or    modified alkylene groups as defined under (a) to (f) hereinbefore,    optionally containing substituents.

Substituents considered are e.g. lower alkyl, e.g. methyl, lower alkoxy,e.g. methoxy, lower acyloxy, e.g. acetoxy, or halogenyl, e.g. chloro.

Further substituents considered are e.g. those obtained when an α-aminoacid is incorporated in the linker R wherein carbon atoms are replacedby amide functions —NH—CO— as defined under (b). In such a linker partof the carbon chain of the alkylene group R is replaced by a group—(NH—CHR′—CO)_(n)— wherein n is between 1 and 100 and R′ represents avarying residue of an α-amino acid.

A further substituent is one which leads to a photocleavable linker R,e.g. an o-nitrophenyl group. In particular this substituento-nitrophenyl is located at a carbon atom adjacent to a amide bond, e.g.in a group —NH—CO—CH₂—CH(o-nitrophenyl)-NH—CO—.

A phenylene group replacing carbon atoms as defined under (e)hereinbefore is e.g. 1,2-, 1,3-, or preferably 1,4-phenylene. Asaturated or unsaturated cycloalkylene group replacing carbon atoms asdefined under (e) hereinbefore is e.g. cyclopentylene or cyclohexylene,or also cyclohexylene being unsaturated e.g. in 1- or in 2-position. Asaturated or unsaturated bicycloalkylene group replacing carbon atoms asdefined under (e) hereinbefore is e.g. bicyclo[2.2.1]heptylene orbicyclo[2.2.2]octylene, optionally unsaturated in 2-position or doublyunsaturated in 2- and 5-position. A heteroaromatic group replacingcarbon atoms as defined under (e) hereinbefore is e.g. triazolidene,preferably 1,4-triazolidene, or isoxazolidene, preferably3,5-isoxazolidene. An saturated or unsaturated heterocyclyl groupreplacing carbon atoms as defined under (e) hereinbefore is e.g.2,5-tetrahydrofuranediyi or 2,5-dioxanediyl, or isoxazolidinene,preferably 3,5-isoxazolidinene.

Preferred linker groups R are, for example, a flexible linker such as aalkyl chain of 1 to 20 carbon atoms, optionally substituted by methyl,methoxy or acetoxy groups, or a polyethylene glycol chain consisting of1 to 20 ethylenoxy groups.

The label part of the substrate can be chosen by those skilled in theart dependent on the application for which the fusion protein isintended. Examples of labels include:

-   (1) A spectroscopic probe such as a fluorophore, a chromophore, a    magnetic probe or a contrast reagent, or also a probe useful in    electron microscopy;-   (2) A radioactively labeled molecule;-   (3) A molecule which is one part of a specific binding pair which is    capable of specifically binding to a partner. Such specific binding    pairs are well known in the art and include, for example, biotin,    which can bind to avidin or streptavidin;-   (4) A molecule that is suspected to interact with other    biomolecules;-   (5) A library of molecules that are suspected to interact with other    biomolecules;-   (6) A molecule which is capable of crosslinking to other    biomolecules as known to those skilled in the art, e.g. as described    by Nadeau et al., 2002;-   (7) A molecule which is capable of generating hydroxyl radicals upon    exposure to H₂O₂ and ascorbate such as a tethered metal-chelate,    e.g. as described by Hod et al., 2002;-   (8) A molecule which is capable of generating reactive radicals upon    irradiation with light such as malachite green, e.g. as described by    Jay et al. 1999;-   (9) A molecule covalently attached to a solid support, where the    support may be a glass slide, a microtiter plate or any polymer in    general known to those proficient in the art;-   (10) A nucleic acid or a derivative thereof capable of undergoing    base-pairing with its complementary strand;-   (11) A lipid or other hydrophobic molecule with membrane-inserting    properties;-   (12) A biomolecule with desirable enzymatic, chemical or physical    properties;-   (13) A molecule possessing a combination of any of the properties    listed above.

The use of a labeled CoA derivative, wherein the phosphopantetheinylmoiety carries a detectable label which can be transferred to the ACPfusion protein, such as a fluorophore, a chromophore, a magnetic probe,a radioactively labeled molecule or any other spectroscopic probe,allows the invention to be used to specifically and covalently attachthe detectable label to the ACP fusion protein, either in vitro or in acell or on the surface of a cell (in vivo). This allows the detectionand characterization of the ACP fusion protein in vivo or in vitro. Theterm in vivo includes labeling in all compartments of a cell as well asof ACP fusion proteins pointing to the extracellular space. The methodcan be compared to the applications of the green fluorescent protein(GFP) which is also genetically fused to the protein of interest andallows its investigation in the living cell. The disadvantage of GFP andits mutants is that it is principally limited to the use of the naturaloccurring fluorophore present in GFP. The labeling inside a cell (invivo) may be used also after fixation of the cell by common fixationprocedures which have only minor impact on the functional structure ofproteins and thus leave the ACP functional with respect to labeling by aphosphopantetheinyl moiety.

The use of a labeled CoA derivative, wherein the phosphopantetheinylmoiety carries an affinity tag such as biotin which can be transferredto the ACP fusion protein, allows the invention to be used to transferan affinity tag to the ACP fusion protein, thereby allowing the fusionprotein to be bound by a binding partner of the affinity tag. By way ofexample, the addition of CoA substrates labeled with an affinity tagsuch as biotin and ACPS to cell extracts (bacterial or eukaryotic)expressing an ACP fusion protein or to purified ACP fusion proteins,will lead to the covalent modification of the fusion protein with theaffinity tag. This will then allow the isolation of the fusion proteinusing the interaction between the affinity tag and its binding partner,e.g. in the case of biotin, with immobilized avidin or streptavidin. Ifthe label is linked to the ACP fusion protein via a linker group Rcontaining a cleavable bond, such as a disulfide bridge, or if thelinker is photocleavable, the ACP fusion protein can be released fromthe affinity tag after its isolation.

The use of a labeled CoA derivative, wherein the phosphopantetheinylmoiety carries a label which can be transferred to the ACP fusionprotein and which is capable of generating reactive radicals, such ashydroxyl radicals, upon exposure to an external stimulus, allows thestudy of conformations of the protein of interest and of proteins in thevicinity. The generated radicals can inactivate the ACP fusion proteinsas well as those proteins that are in close proximity of the ACP fusionprotein, allowing the study the role of these proteins. Examples of suchlabels are tethered metal-chelate complexes that produce hydroxylradicals upon exposure to H₂O₂ and ascorbate, and chromophores such asmalachite green that produce hydroxyl radicals upon laser irradiation.The use of chromophores and lasers to generate hydroxyl radicals is alsoknown in the art as chromophore assisted laser induced inactivation(CALI), for example as described by Jay et al., 1998. CALI is a methodthat is used to specifically inactivate certain proteins within a cellin a time-controlled and spatially-resolved manner and which is basedupon the spatial neighbourhood of a chromophore and a protein. Uponlaser irradiation the chromophore generates hydroxyl radicals, whichinactivate all proteins within and only within about 100 nm of thechromophore. So far, the chromophore is brought in the spatialneighbourhood of the protein of interest by microinjectingchromophore-labeled antibodies specific to the protein of interest. Inthe present invention, labeling ACP fusion proteins with chromophoressuch as malachite green and subsequent laser irradiation would allow toinactivate the ACP fusion protein as well as those proteins thatinteract with the ACP fusion protein in a time-controlled andspatially-resolved manner. The method can be applied both in vivo or invitro.

In a similar manner, ACP fusion proteins can be labeled with tetheredmetal-chelates and the ACP fusion protein and those proteins thatinteract with the ACP fusion protein can be inactivated in a specificmanner upon exposure to H₂O₂ and ascorbate. The method can not only beused to study the function of an ACP fusion protein or those that are inclose proximity of the ACP fusion protein, but also to identify thoseproteins that are in close proximity of a ACP fusion protein. Here,proteins which are in close proximity of the ACP fusion protein can beidentified as such by either detecting fragments of that protein by aspecific antibody, by the disappearance of those proteins on ahigh-resolution 2D-electrophoresis gels or by identification of thecleaved protein fragments via separation and sequencing techniques suchas mass spectrometry or protein sequencing by N-terminal degradation.

The use of a labeled CoA derivative, wherein the phosphopantetheinylmoiety carries a ligand which can be transferred to the ACP fusionprotein, allows the invention to be used to specifically attach theligands and binding partners of the ligand, such as proteins, to the ACPfusion protein. If the ligand binds to another protein Y and thedimerization of the protein Y with the labeled ACP fusion protein leadsto a biological function or a measurable signal, the biological functionor the measured signal depends on the addition of the labeled ACP fusionprotein. If ACP is coupled to proteins displayed on cell surfaces theinteraction with the ligand can mediate contacts with other moleculesmodified by the ligand, including biomolecules, either individually oras part of other cells, of tissue, and of intact organisms modified bythe ligand.

The use of a labeled CoA derivative, wherein the phosphopantetheinylmoiety is covalently attached to the surface of a carrier, or whereinthe label is a molecule that can be bound non-covalently by anothermolecule that is itself attached to the surface, allows the invention tobe used to construct protein arrays on a solid support. An example forthe latter approach is where the label is biotin and the moleculeattached to the surface is streptavidin or avidin. Possible examples fora carrier are a glass side, a microtiter plate or any functionalizedpolymer. The CoA derivative used as a substrate is immobilized on thecarrier via its label, and the subsequent ACPS-catalyzed reaction of anACP fusion protein immobilizes such fusion protein on the carrier by thetransfer of the label to the fusion protein. Spotting (different) ACPfusion proteins together with ACPS in a spatially resolved manner on thecarrier pretreated with the corresponding CoA derivative allows thecreation of protein arrays.

The use of a labeled CoA derivative, wherein the phosphopantetheinylmoiety is covalently attached to a label which is a molecule that cancross-link to other proteins, allows the invention to be used to studyinteractions of the protein of interest in a suitable environment.Examples of such cross-linkers are molecules containing functionalgroups such as maleimides, active esters or azides, and others known tothose proficient in the art, e.g. those described in Nadeau et al.,2002. Contacting such labeled CoA derivatives in the presence of ACPSwith ACP fusion proteins that interact with other proteins (in vivo orin vitro) can lead to the covalent cross-linking of the ACP fusionprotein with its interacting protein via the label. This allows theidentification of the protein interacting with the ACP fusion protein.

The use of a labeled CoA derivative, wherein the phosphopantetheinylmoiety carries a ligand which can be transferred to an ACP fusionprotein consisting of ACP and a membrane receptor and which allowsdetection of the labeled receptor. This labeling of the cell surfacereceptor allows to observe receptor internalization. A particularapplication is the investigation of the internalization of receptorproteins after binding of a ligand, for example by a drug, a drug leador a tentative drug lead. The receptor internalization can be detectedby various methods, e.g. by microscopic detection of translocation ofthe label from the cell membrane to the cell interior or by changes inthe fluorescence characteristics of the label upon transfer to theintracellular milieu. Such changes can also be detected withoutmicroscopic tools and may be facilitated by the addition of a quencherto the extracellular medium.

The use of a labeled CoA derivative, wherein the phosphopantetheinylmoiety carries a ligand which can be directly detected by electronmicroscopy, e.g. electron dense nanoparticles. Another application ofthe phosphopantetheinyl moiety for electron microscopy detection isbased on labels used as photosensitizers such as eosin to oxidize uponillumination aromatic amines, such as dianisidine, into an electrondense precipitate which can then be detected by electron microscopy.

By way of example, embodiments of the present invention will now bedescribed in more detail with reference to the accompanying figures.

EXAMPLES

The following examples and experimental procedures are set forth so asto provide those of ordinary skill in the art with a complete disclosureand description of how to practice the invention, and are not intendedto limit the scope of the invention.

Synthesis of CoA-Bt

To a solution of biotin-maleimide 1 (1 mg, 0.0022 mmol) in 100 μl DMF, asolution of coenzyme A disodium salt (1.79 mg, 0.0022 mmol, 1 eq.) in 90μl DMF and 10 μl 50 mM Tris-Cl pH 7.5 is added. The mixture is stirredfor 4 hours at room temperature. It is then diluted with CH₃CN/H₂O 1:4,and aliquots of 500 μl are injected on a preparative HPLC column:Gradient (A=H₂O 99%, CH₃CN 1%, 50 mM NH₄OAc/B=CH₃CN) from A/B 95:5 toA/B 80:20 in 2 min, to A/B 68:32 in 7 min, to A/B 20:80 in 2 min, thenback to A/B 95:5. The retention time of the CoA-biotin is 6.5 min.Fractions containing the desired product are concentrated in vacuo,dissolved in DMSO, and an analytical amount injected to control thepurity. The pure fractions are combined. The concentration of CoA-biotinis determined by absorption at 260 nm (ε(adenine, 260 nm)=15'300 [M⁻¹cm⁻¹]). The yield of CoA-Bt is 0.895 mg (33%).

ESI-MS (m/z) calculated 1217.296 [M(−1)], found 1217.2657 [M(−1)]

Synthesis of CoA-Dg

To a solution of N-(ε-maleimidocaproic acid) hydrazide 2 (1 mg, 0.0044mmol) in 50 μl DMF, a solution of coenzyme A disodium salt (3.6 mg,0.0044 mmol, 1 eq.) in a DMF/buffer mixture (70 μl DMF/30 μl 50 mM TrisCl, pH 7.5) is added. The reaction is followed by analytical HPLC(detection at 260 nm) to verify the completion of the reaction.Subsequently, a solution of 3-amino-3-deoxydigoxigenin hemisuccinamidesuccinimidyl ester (2.6 mg, 0.0044 mmol, 1 eq.) dissolved in 50 μl DMFand 10 μl Et₃N is added, and the reaction mixture stirred for 4 hours atroom temperature. The reaction is submitted to preparative HPLC andfractions containing the desired product are concentrated in vacuo,dissolved in DMSO, and analyzed by analytical HPLC for purity. Fractionscontaining pure product are combined. The concentration of CoA-Dg isdetermined using the extinction coefficient of adenine (ε(adenine, 260nm)=15′300 [M⁻¹ cm⁻¹]). The yield of CoA-Dg is 2.37 mg (37%).

ESI-MS (m/z) calculated 1462.3707 [M(−1)], found 1462.481 [M(−1)].

Synthesis of CoA-Cy3

To a solution of Cy3-maleimide 3 (Pharmacia, 1 mg, 0.00126 mmol) in 100μl DMF, a solution of coenzyme A disodium salt (1.05 mg, 0.00126 mmol, 1eq.) in 90 μl DMF and 10 μl 50 mM Tris.Cl pH 7.5 is added. The mixtureis stirred for 4 hours at room temperature. It is then diluted withCH₃CN/H₂O 1:4, and aliquots of 500 μl are injected on a preparative HPLCcolumn: Gradient from NB 95:5 to NB 90:10 in 2 min, to A/B 65:35 in 15min, to NB 20:80 in 2 min, then back to A/B 95:5 (A and B see precedingexample). The retention time of CoA-Cy3 is 10 min. Fractions containingthe desired product are concentrated in vacuo, dissolved in DMSO, and ananalytical amount injected to control the purity. The pure fractions arecombined. The concentration of CoA-Cy3 is determined by absorption at549 nm (ε (549 nm)=150′000 [M⁻¹ cm⁻¹]). The yield of CoA-Cy3 is 0.847 mg(44%).

ESI-MS (m/z) calculated 1519.370 [M(−1)], found 1519.3071 [M(−1)].

Synthesis of CoA-Cy5

The synthesis of CoA-Cy5 starting with the Cy5-maleimide 4 (1 mg,0.00122 mmol) is performed as described for CoA-Cy3. The concentrationof CoA-Cy5 is determined using the extinction coefficient of Cy5 (ε (646nm)=250′000 [M⁻¹ cm⁻¹]). The yield of CoA-Cy5 is 0.828 mg (44%).

ESI-MS (m/z) calculated 1545.386 [M(−1)] and 772.189 [M(−2)], found771.6524 [M(−2)].

Cloning, Expression and Purification of 6xHis-ACPS

The XL1-blue E. coli ACPS gene is amplified by single colony PCR andcloned in a pET-15b plasmid (Novagen) using the forward primer 5′-TCTGGT CAT ATG GCA ATA TTA GGT TTA GGC ACG G-3′ with the NdeI restrictionsite (underlined), and the backward primer 5′-TCA AGT CTC GAG TTA ACTTTC AAT AAT TAC CGT GGC A-3′ with the XhoI restriction site(underlined). The sequence of the peptide (originating from the plasmidpET-15b) fused to the N-terminus of E. coli ACPS is (in single lettercode) MGSSHHHHHHSSGLVPRGSH followed by the first amino acid of ACPS, amethionine. This fusion protein is designated 6xHis-ACPS.

Liquid cultures of BL21 E. coli cells containing a pET-15b (Novagen)based expression vector encoding 6xHis-ACPS are grown to an opticaldensity OD_(600nm) of 0.6. Expression of 6xHis-ACPS is induced by addingIPTG to a final concentration of 1 mM. After incubation for 3.5 hours at220 rpm at 24° C., the culture is centrifuged for 10 minutes at 3000 gat 4° C. The pellet is resuspended in 10 ml extract-buffer (150 mM NaCl,5 mM imidazole, 50 mM KH₂PO₄, pH 8.0), and PMSF and aprotimine are addedto a final concentration of 1 mM and 2 μg/ml respectively. Lysozyme isadded to 1 ring/ml, and the mixture is incubated for 15 minutes on iceand inverted several times. Then it is sonicated for 10 minutes (95%Power, 50% Duty). DNase I is added to a final concentration of 0.01mg/ml. After 30 minutes at 4° C., the mixture is centrifuged for 10minutes at 18′000 rpm.

For the purification of the protein, 350 μl of Ni-NTA, previously washedthree times with extract buffer, are added to the lysate. The mixture isincubated 20 minutes on ice and mixed several times. The extract mixtureis then added to a polypropylene column, which is allowed to drain. Thecolumn is washed with 5×400 μl DNA elution buffer (10 mM Tris.Cl, pH8.5), then with 2×5 ml wash buffer (300 mM NaCl, 10 mM imidazole, 50 mMKH₂PO₄, pH 7.5). To elute the protein, elution buffer (300 mM NaCl, 150mM imidazole, pH 7.5) is added to the column, incubated for 10 minutes,and then the flow-through is collected. Elution is continued stepwisewith 150 μl elution buffer until no more protein is detectable in aBradford assay. Finally the combined eluates are dialysed overnight indialysis buffer (50 mM HEPES, 30% glycerol, pH 7.2) to remove theresidual salts. The pure 6xHis-ACPS (MW 16.215 kDa) is aliquoted andstored at −80° C. The concentration is determined by a Bradford assay tobe 37.6 μM. Yield 0.912 mg per liter of BL21 E. coli cell culture.

Cloning, Expression and Purification of 6xHis-ACP

The XL1-blue E. coli ACP gene is amplified by single colony PCR andcloned in a pET-15b plasmid (Novagen) using the forward primer 5′-GT CGGTAT CAT ATG AGC ACT ATC GAA GAA CG-3′ with the NdeI restriction site(underlined) and the backward primer 5′-TCA TGC GGA TCC TTA CGC CTG GTGGCC GTT G-3′ with the BamHI restriction site (underlined). The sequenceof the peptide fused to the N-terminus of E. coli ACP (yielding6xHis-ACP) is (in single letter code) MGSSHHHHHHSSGLVPRGSH followed bythe first amino acid of ACP, a methionine.

Liquid cultures of BL21 E. coli cells containing a pET-15b (Novagen)based expression vector encoding 6xHis-ACP are grown to an opticaldensity OD_(600nm) of 0.6. Expression of 6xHis-ACP is induced by addingIPTG to a final concentration of 1 mM. After incubation for 3.5 hours at220 rpm at 37° C., the culture is centrifuged for 10 minutes at 3000 gat 4° C. The pellet is treated exactly as described in the aboveexperiment for the purification of 6xHis-ACPS. The concentration of6xHis-ACP (MW 10.802 kDa) is determined by a Bradford assay to be 188μM. Yield: 3.1 mg per liter of BL21 E. coli cell culture. To determinethe ratio between apo- and holo-6xHis-ACP, purified 6xHis-ACP isanalyzed by ESI-MS (pos. mode) using a Q-T of-Ultima (Micromass/Waters),optionally coupled to Cap-LC (Waters), chromatography on Xterra RP-C4column (Waters, 5 μm, 0.32×50 mm; flow 8 μl/min) and deconvolution byMaxEnt1-software. ESI-MS of the mixture demonstrates that thepreparation contains a mixture of holo- and apo-ACP. The mass ofpurified apo-6xHis-ACP without the first methionine is found to be10.6720 kDa (calculated 10.6716 kDa), and that of holo-6xHis ACP to be11.012 kDa (calculated 11.0119 kDa). LC-ESI-MS allows the identificationand integration of the peaks corresponding to the holo- and apo-form.The percentage of the holo-form is determined to be 16% (retention time11.77 min), and that of the apo-form to be 84% (retention time 14.16min).

Expression and Purification of 6xHis-ACP-ha

The sequence of the peptide fused to the N-terminus of E. coli ACP is(in single letter code) MGSSHHHHHHSSGLVPRGSH followed by the first aminoacid of ACP, a methionine, and the sequence of the peptide fused to theC-terminus of E. coli ACP is (in single letter code) TSRSYPYDVPDYARW(yielding 6xHis-ACP-ha).

Liquid cultures of BL21 (DE3) E. coli cells containing a pET-15b basedexpression vector encoding 6xHis-ACP-ha are grown to an optical densityOD_(600nm) of 0.6. Expression of 6xHis-ACP-ha is induced by adding IPTGto a final concentration of 1 mM. After incubation for 3 hours at 220rpm at 24° C., the culture is centrifuged for 10 minutes at 3000 g at 4°C. 6xHis-ACP is then purified as described for the purification of6xHis-AcpS. The concentration of 6xHis-ACP-ha (MW 12.65 kDa) isdetermined by Bradford assay to be 400 μM, and the total yield ofprotein is 10 mg per liter of shake-flask culture.

In Vitro Biotinylation of 6xHis-ACP Using CoA-Bt and 6xHis-ACPS

Purified 6xHis-ACP (1 μM) is incubated with 6xHis-ACPS (0.2 μM) andMgCl₂ (10 mM) in reaction buffer (43 μl, 50 mM Tris Cl, pH 8.8) at roomtemperature. An aliquot of 7.5 μl is taken for analysis. CoA-Bt is addedto a final concentration of 5 μM. Aliquots of 7.5 μl are taken atdefined times. The aliquots are quenched for 30 seconds with coenzyme A(1 mM final concentration), and 8.2 μl SDS-buffer 2× are added. Thesamples are heated for 2 minutes at 95° C. The biotinylated 6xHis-ACP isdetected by Western-blotting, using a streptavidin-horseradishperoxidase conjugate (NEN) and a chemiluminescent peroxidase substrate(Renaissance reagent plus, NEN). The western blot is analyzed using animage station (Kodak 440). As controls, samples containing each proteinalone, and samples containing only one of the two proteins and CoA-Btare also prepared and analyzed for biotinylation as above in order tocheck the background and to check the specificity of the reaction. Thebiotinylation depends on the presence of all three components.

Quantification of Biotinylation of 6xHis-ACP via Gel Shift Assay

Purified 6xHis-ACP (3 μM) is incubated at RT with CoA-Bt (10 μM) andpurified 6xHis-ACPS (5 μM) in a final volume of 50 μl reaction buffer(50 mM Tris.Cl, pH 8.8, 10 mM MgCl₂). After 30 minutes of incubation,the mixture is dialyzed overnight against TBS (10 mM Tris.Cl, 150 mMNaCl, pH 7.9) to remove any excess CoA-Bt. Aliquots of the reaction areincubated with streptavidin for 1 h at a final concentration of 0.6μg/μl. 2×SDS sample buffer containing only 2% SDS is added to samples,and SDS-PAGE is performed directly without heating the sample. Proteinsare detected by Coomassie staining, and the degree of biotinylation isestimated by comparison of the band intensities with samples containingthe identical amount of 6xHis-ACP not biotinylated and not incubatedwith streptavidin.

Competition Assays between CoA and CoA-label

Purified 6xHis-AcpS (0.4 μM) is incubated at RT with either CoA-Bt orCoA-Dg (2 μM), variable amounts of CoA (0, 2, 4, 8, 12, 20, 40, 80 μM)and purified 6xHis-ACP-ha (0.4 μM) in 20 μl reaction buffer (50 mMTris.Cl, pH 7.5, 10 mM MgCl₂). After 25 minutes, each sample is quenchedby addition of 20 μl of 2×SDS sample buffer and heated at 95° C. for 2minutes. Labeled 6xHis-ACP-ha is detected by Western-blotting usingeither a streptavidin-horseradish peroxidase conjugate (dilution1:12500) or an anti-digoxigenin antibody-horseradish peroxidaseconjugate (dilution 1:500) and a chemiluminescent peroxidase substrate.Signal intensities at 0 μM CoA are arbitrary set to 1 for eachexperiment.

Labeling of Aga2-ACP on the Surface of Yeast Cells

The sequence of AGA2 is amplified from yeast genomic DNA and inserted inthe yeast expression vector pRS314 behind the P_(CUP1)-promoter sequenceusing the EcoRI and SalI restriction sites introduced at the ends of thePCR fragment. ACP is cloned in frame behind AGA2 using a SalI and anAcc651 restriction site introduced by the PCR amplification of the ACPsequence. ACP is extended by a sequence encoding the HA-epitope. Thesequence connecting Aga2p and ACP reads: FVDEMLYFQGM. The last residueof Aga2p and the first residue of ACP are underlined. The C-terminalsequence of ACP including the HA-epitope reads: QAYPYDVPDYAG. The lastresidue of ACP is underlined. Yeast strain EBY100 (MATa ura3-52 trp1leu2Δ1 his3Δ200 pep4::HIS3 can1 GAL pIU211:URA3) (Invitrogen, Carlsbad,Calif.) expressing Aga1p from the P_(GAL1)-promoter and Aga2-ACP fromthe P_(CUP1)-promoter are grown in 10 ml of selective medium containing2% galactose and 0.1 mM copper to an OD₆₀₀ of 1.4 OD₆₀₀ units of thecells are washed with 2 ml water and resuspended in 0.2 ml labelingbuffer (50 mM Tris Cl pH 8.8, 100 mM NaCl, 10 mM MgCl₂). CoA substrateand 6xHis-ACPS are added to a final concentration of 10 and 1 μM,respectively. Labeling is stopped after 20 min at RT by diluting thereaction into 2 ml PBS. The cells are washed four times with 2 ml of PBSand either subjected to fluorescence microscopy directly or after a 20min incubation in 0.2 ml of Qdot™incubation buffer containing 20 nMQdot™605 streptavidin conjugate (Milan Analytica AG, Switzerland)followed by washing the cells in four times 2 ml of PBS. Cells areinspected with a Zeiss Axiovert 135 fluorescence microscope (Carl Zeiss,Göttingen, Germany) using a 63× oil (1.4 numerical aperture) objective.

Labeling of ACP-NK1 Fusion Protein Displayed on HEK293 Cells

For the transient expression of ACP-NK1, the signal sequence of the5-HT₃-receptor (Sig_(5HT3)) is fused to the N terminus of ACP via ashort DYV linker and NK1 is fused to the C terminus of ACP via a shortTS linker. In the resulting construct, a FLAG tag and a 6xHis tag isalso attached to the C terminus of NK1. The corresponding gene of thefusion protein is inserted into the NheI and BamHI sites of the vectorpCEP4 (Invitrogen). HEK293 cells are grown in DMEM/F2 (Dulbecco'smodified Eagle medium, GIBCO BRL) supplemented with 2:2% fetal calfserum (GIBCO, BRL). Transient transfection is performed as described(Nat Biotechnol 21, 86-89 (2003)) and the HEK293 cells areco-transfected with vectors expressing ACP-NK1 and a nuclear targetedEGEP (EGFP-NLS₃). After 24 h, cells are incubated for 10 min at roomtemperature with 500 μl of PBS buffer containing MgCl₂ (10 mM),6xHis-AcpS (1 μM) and either CoA-Cy3, CoA-Cy5 or CoA-Bt (each 5 μM). Thecells are then washed three times with PBS to remove any excesssubstrate and directly analyzed by laser-scanning confocal fluorescencemicroscopy when labeled with Cy3 or Cy5. Biotinylated cells areincubated with FluoroLink™ Cy5-labeled streptavidin (AmershamBiosciences) at concentrations of 1 μg/ml in PBS before being washedthree times with PBS. Laser-scanning confocal micographs are recordedusing a 488 nm argon/krypton laser line; a 543 nm HeNe laser line or a633 nm HeNe laser line on a Zeiss LSM 510 microscope (Carl Zeiss AG,Göttingen, Germany) with a 63× water (1.2 numerical aperture) objective.Scanning speed and laser intensity are adjusted to avoid photobleachingof the fluorescent probes, and damage or morphological changes of thecells. Fluorescence is analyzed in the channels sensitive to GFP andsensitive to the respective dye. For each cell sample tested a clearnuclear labeling is observed in the GFP channel, and a clear membranelabelling is observed in the cell membrane region after labelling withCoA-Cy3, after labelling with CoA-Cy5, and after labelling with CoA-Btand subsequent staining with Streptavidin-Cy5. No membrane staining isobserved for non-transfected cells showing no nuclear expression of GFP,indicating a specific labeling of ACP-NK1 with Cy3, Cy5 and biotin,respectively. The ACP-NK1 receptor transiently expressed in HE293 cellsis co-stained in a further experiment with CoA-Cy5 and withtetramethylrhodamine-labeled substance P(SP-rho), the natural ligand ofNK₁. Both substance stain exclusively the membrane area and lead toidentical stained regions. The reversal of the SP-rho staining by anexcess of unlabeled substance P within one minute furthermore proves thespecificity of the labelling and also the functionality of ACP-NK1 withrespect to ligand binding.

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The invention claimed is:
 1. A labeled CoA type substrate of the formula(I) or (II):

wherein R is a linker group bridging the coenzyme A and the label; andwherein R is a straight or branched chain alkylene group with 1 to 300carbon atoms, wherein (a) R has at least 3 carbon atoms wherein everythird carbon atom is replaced by oxygen; or (b) one or more carbon atomsare replaced by nitrogen carrying a hydrogen atom, and the adjacentcarbon atoms are substituted by oxo, representing an amide function—NH—CO—; or (c) one or more carbon atoms are replaced by oxygen, and theadjacent carbon atoms are substituted by oxo, representing an esterfunction —O—CO—; or (d) the bond between two adjacent carbon atoms is adouble or a triple bond, representing a function —CH═CH— or —C≡C—; or(e) one or more carbon atoms are replaced by a phenylene, a saturated orunsaturated cycloalkylene, a saturated or unsaturated bicycloakylene, abridging heteraromatic or a bridging saturated or unsaturatedheterocyclyl group; or (f) two adjacent carbon atoms are replaced by adisulfide linkage —S—S—; or a combination of two or more, especiallytwo, alkylene and/or modified alkylene groups as defined under (a) to(f) hereinbefore, optionally containing substituents; and “Label” is alabel molecule suitable for the detection and/or manipulation of afusion protein in a system designed for detecting and/or manipulatingthe label.
 2. A labeled CoA type substrate according to claim 1 wherein“Label” is selected from the group consisting of (1) a spectroscopicprobe; (2) a radioactively labeled molecule; (3) a molecule which is onepart of a specific binding pair which is capable of specifically bindingto a partner; (4) a molecule that is suspected to interact with otherbiomolecules; (5) a library of molecules that are suspected to interactwith other biomolecules; (6) a molecule which is capable of crosslinkingto other biomolecules; (7) a molecule which is capable of generatinghydroxyl radicals upon exposure to H₂O₂ and ascorbate; (8) a moleculewhich is capable of generating reactive radicals upon irradiation withlight; (9) a molecule covalently attached to a solid support; (10) anucleic acid or a derivative thereof capable of undergoing base-pairingwith its complementary strand; (11) a lipid or other hydrophobicmolecule with membrane-inserting properties; (12) a biomolecule withdesirable enzymatic, chemical or physical properties; and (13) amolecule possessing a combination of any of the properties (1) to (12).3. A labeled CoA type substrate according to claim 1 wherein “Label” isselected from biotin, digoxigenin, Cy-3 and Cy-5.
 4. A method fordetecting and/or manipulating a protein of interest using a labeled CoAtype substrate according to claim 1, which comprises: (a) contacting afusion protein comprising (i) a protein of interest consisting of atleast 12 amino acids and up to 2,000 amino acids and (ii) an acylcarrier protein (ACP) or a fragment thereof containing a serine residueto which a phosphopantetheine moiety of a labeled coenzyme A (Co A) typesubstrate according to claim 1 is attachable; by means of a holo-acylcarrier protein synthase (ACPS) or a homologue thereof selected from thegroup consisting of EntD, Sfp, Psf-1, Gsp, LYS5, Bli, Lpal4, and NshC,(b) transferring the label from the CoA type substrate to the fusionprotein in the presence of ACPS; and (c) detecting and/or furthermanipulating the labeled fusion protein obtained, using the label in asystem designed for detecting and/or manipulating the label.